In recent years, liquid chromatography in combination with mass spectrometry (LC-MS) and in particular liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS) has become a common technique for the analysis of phosphorylated proteins. Typically a protein sample is digested using a proteolic enzyme, such as trypsin. From the digest mixture, phosphopeptides are separated via liquid chromatography and then analysed via tandem mass spectrometry.
The most significant problems with the above-described method are the relative sample complexity, particularly when analysing whole cells, and the low concentrations of phosphopeptides of such samples. Without implementing specific enrichment or extraction steps, it is only possible to identify the most abundant phosphopeptides.
A successful method of phosphopeptide enrichment in the art is the use of immobilised metal ion affinity chromatography (IMAC). The IMAC technique relies upon the differing affinities of some peptides for particular metal ion complexes. Though this method does give some improvements in concentration of phosphopeptides, samples still show contamination with other peptides.
The term “peptide” as used herein, refers to a polymeric chain of two or more amino acids, each linked by an amide group represented by the formula —COONRa—, where Ra refers to hydrogen or any possible side-chain. A peptide may also include a number of modifications, including phosphorylation, lipidation, prenylation, sulfation, hydroxylation, acetylation, addition of carbohydrate, addition of prosthetic groups or cofactors, formation of disulfide bonds, proteolysis, assembly into macromolecular complexes and the like.
The term “phosphopeptide” as used herein, refers to the phosphorylated form of a peptide, where a phosphate group (PO4)3− is added to the chain at one or more of the amino acid groups, giving an increase in mass corresponding to HPO3.
The term “protein” as used herein, refers to a polymeric chain of peptides. A protein may also include a number of modifications, including phosphorylation, lipidation, prenylation, sulfation, hydroxylation, acetylation, addition of carbohydrate, addition of prosthetic groups or cofactors, formation of disulfide bonds, proteolysis, assembly into macromolecular complexes and the like.
The term “phosphoprotein” as used herein, refers to the phosphorylated form of a protein, where a phosphate group (PO4)2− is added to the chain at one or more of the amino acid groups.
The term “glycoprotein” is used to denote a protein linked to a saccharide group and a glycopeptide is a peptide linked to a saccharide group.
The term “eluent” as used herein, refers to the mobile phase in a chromatographic separation. Such a mobile phase may be a single organic solvent, water, an aqueous ionic solution, a mixture of organic solvents, a mixture of organic solvents and water or a mixture of organic solvents and aqueous ionic solution. The makeup or concentration of an eluent may change or be changed during the course of a chromatographic separation.
The term “counterion” as used herein, refers to an ion in solution in an eluent, which may displace other ions bound to the stationary phase in a chromatographic separation.
The term “eluate” as used herein, refers to a combination of the eluent and solute exiting the stationary phase after chromatographic separation.
It is an object of the present invention to provide an improved method for the separation, isolation or enrichment of analytes from a sample solution. The separated, isolated or enriched analyte may then be stored or analysed as required.